The overall objective of this proposal is to define the cellular mechanisms contributing to carcinogenesis of cholangiocytes, the epithelia cells lining the biliary system. Chronic inflammation of the biliary tree predisposes patients to the development of cholangiocarcinoma. Because of this relationship, our long term goal is to understand the mechanisms by which inflammatory mediators contribute to the development and progression of cholangiocarcinoma. We have recently generated striking preliminary data demonstrating that cholangiocarcinomas paradoxically express the potent apoptosis inducing ligand TRAIL. However, because the inflammatory mediator IL-6 upregulates the anti-apoptotic protein Mcl-1, they are resistant to TRAIL cytotoxicity. These same characteristics, paradoxical TRAIL expression and upregulation of Mcl-1, were also identified in a newly described genetic murine model of cholangiocarcinoma. Based on these extensive preliminary data, we propose the central hypothesis that the inflammatory cytokine IL-6 upregulates Mcl-1, which in turn circumvents TRAIL cytotoxicity. Our proposal has three specific aims. First, we will test the hypothesis that IL-6 upregulates Mcl-1 by both transcriptional and post-translational mechanisms due to SOCS3 silencing and Akt-mediated Mcl-1 phosphorylation, respectively. Second, we will test the hypothesis that Mcl-1 inhibits TRAIL-mediated apoptosis by binding specific pro-apoptotic BH3- only proteins of the Bcl-2 family, thereby, blocking the lysosomal pathway of cell death. Finally, we will test the hypothesis that Mcl-1 expression and function can be inhibited in a murine model of cholangiocarcinoma, resulting in tumor autonomous, TRAIL-mediated apoptosis. To address these questions, we have established cholangiocarcinoma cell lines that differentially express Mcl-1 and have become adept at dissecting IL-6 and TRAIL signaling pathways. We have also developed an understanding of Mcl-1 structure and function relationships. Finally, we have developed a collaboration to test the proposed concepts in a recently described murine model of cholangiocarcinoma. The proposal is conceptually and technically innovative as it tests new concepts as to how inflammatory mediators promote a major cancer phenotype, evasion of apoptosis. The information emanating from these studies may potentially help identify therapeutic strategies for the treatment and/or chemoprevention of cholangiocarcinoma.